U.S. patent application number 12/816999 was filed with the patent office on 2011-12-22 for methods of spraying saccharification enzymes and fermentation organisms onto lignocellulosic biomass for hydrolysis and fermentation processes.
Invention is credited to Dwight ANDERSON, Johnway GAO, Benjamin LEVIE.
Application Number | 20110312033 12/816999 |
Document ID | / |
Family ID | 45327285 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110312033 |
Kind Code |
A1 |
GAO; Johnway ; et
al. |
December 22, 2011 |
METHODS OF SPRAYING SACCHARIFICATION ENZYMES AND FERMENTATION
ORGANISMS ONTO LIGNOCELLULOSIC BIOMASS FOR HYDROLYSIS AND
FERMENTATION PROCESSES
Abstract
The present invention provides spray methods of delivering
saccharification enzymes, fermentation organisms, and other
hydrolysis or fermentation ingredients onto lignocellulosic
biomass. The methods reduce the need for mechanical mixing when the
biomass solids are undergoing enzymatic hydrolysis, and reduce
dilution to allow higher product titers in the hydrolysis and/or
fermentation steps.
Inventors: |
GAO; Johnway; (Federal Way,
WA) ; LEVIE; Benjamin; (Mercer Island, WA) ;
ANDERSON; Dwight; (Puyallup, WA) |
Family ID: |
45327285 |
Appl. No.: |
12/816999 |
Filed: |
June 16, 2010 |
Current U.S.
Class: |
435/72 |
Current CPC
Class: |
Y02E 50/16 20130101;
C12P 19/14 20130101; Y02E 50/10 20130101; C13K 1/02 20130101; C12P
19/02 20130101; C12P 7/10 20130101 |
Class at
Publication: |
435/72 |
International
Class: |
C12P 19/00 20060101
C12P019/00 |
Claims
1. A method of producing a saccharification product from a
pretreated biomass composition, the method comprising: a) spraying
a liquid comprising one or more saccharification enzymes onto the
pretreated biomass composition to form an impregnated biomass
composition; and b) maintaining the impregnated biomass composition
under suitable conditions to promote hydrolysis of components in
the impregnated biomass composition to produce the saccharification
product.
2. The method of claim 1, wherein the liquid further comprises one
or more fermentation organisms, and wherein the one or more
fermentation organisms ferment sugars produced by the hydrolysis to
produce a fermentation product.
3. A method of producing a saccharification product from a
pretreated biomass composition comprising a prehydrolysate and
pretreated biomass solids, the method comprising: a) separating the
prehydrolysate from the pretreated biomass solids to form a first
prehydrolysate liquid; b) adding one or more saccharification
enzymes to the first prehydrolysate liquid to form a second
prehydrolysate liquid; c) spraying the second prehydrolysate liquid
onto the pretreated biomass solids to form an impregnated biomass
composition; and d) maintaining the impregnated biomass composition
under suitable conditions to promote enzymatic hydrolysis of the
impregnated biomass to produce the saccharification product.
4. The method of claim 3, wherein the method further comprises: a)
adding one or more fermentation organisms to the saccharification
product; and b) maintaining the saccharification product under
suitable conditions to promote fermentation of sugars produced by
hydrolysis to produce a fermentation product.
5. The method of claim 3, wherein the pretreated biomass solids are
not rinsed or conditioned before spraying with the second
prehydrolysate.
6. The method of claim 3, further comprising adjusting the pH of
the first or second prehydrolysate to a pH range of 3.0 to 9.0
prior to the spraying step.
7. The method of claim 1, wherein the one or more saccharification
enzymes are selected from the group consisting of cellulase,
beta-glucosidase, xylanase, other hemicellulases, and mixtures
thereof.
8. The method of claim 2, wherein the fermentation organisms are
selected from the group consisting of yeast, mold, algae, bacteria,
and mixtures thereof.
9. The method of claim 3, further comprising mixing the pretreated
biomass composition with the second prehydrolysate liquid.
10. The method of claim 1, wherein the pretreated biomass solids
are transported on a screw or a belt conveyer during the spraying
step to form the impregnated biomass composition.
11. The method of claim 1, further comprising moving the
impregnated biomass composition with a belt or screw conveyer into
a reactor for the hydrolysis.
12. The method of claim 1, wherein the pretreated biomass
composition comprises wood chips, and wherein the wood chips are
size reduced prior to pretreatment, such that prior to size
reduction 50-100% of the wood chips pass through a +7 mm round hole
screen, and after size reduction, 50-100% of the wood chips pass
through a +3 mm round hole screen.
13. The method of claim 3, wherein the pretreated biomass solids
are ground up prior to spraying with the second prehydrolysate
liquid.
14. The method of claim 3, wherein the pretreated biomass
composition is produced from an acid sulfite or acid bisulfite
pretreatment process.
15. The method of claim 3, wherein the pretreated biomass
composition is produced from an alkaline sulfite or alkaline
bisulfite pretreatment process.
16. The method of claim 3, wherein the pretreated biomass
composition is produced from a neutral chemical pretreatment
process or a pretreatment process using water and no additional
chemicals.
17. The method of claim 2, wherein the fermentation product has a
titer greater than 4.0%.
18. The method of claim 3, wherein the hydrolysis produces a sugar
yield of 80-100% of the pretreated biomass composition.
19. The method of claim 2, wherein the fermentation converts
60-100% of the sugars to the fermentation product.
20. A method of producing a fermentation product from a pretreated
biomass composition comprising a prehydrolysate and pretreated
biomass solids, the method comprising: a) separating the
prehydrolysate from the pretreated biomass solids to form a first
prehydrolysate liquid; b) adding one or more saccharification
enzymes and one or more fermentation organisms to the first
prehydrolysate liquid to form a second prehydrolysate liquid; c)
spraying the second prehydrolysate liquid onto the pretreated
biomass solids to form an impregnated biomass composition; and d)
maintaining the impregnated biomass composition under suitable
conditions to promote enzymatic hydrolysis of the impregnated
biomass composition and fermentation of the saccharification
product to form the fermentation product.
Description
BACKGROUND
[0001] 1. Field
[0002] The present disclosure relates generally to spray methods
for delivering saccharification enzymes and fermentation organisms
onto lignocellulosic biomass. More specifically it relates to spray
methods using a pretreatment prehydrolysate as the delivery liquid
in hydrolysis or simultaneous hydrolysis and fermentation
processes, thereby reducing the amount of dilution and the need to
mix the saccharification or fermentation reaction mixture, while
increasing the titer of a saccharification and/or fermentation
product.
[0003] 2. Related Art
[0004] In the hydrolysis and fermentation of cellulosic biomass to
ethanol or other biofuels and bioproducts, the titer of the final
product is lower than starch based fermentation, and therefore
requires more energy for distillation. A minimum solids loading of
10% going into hydrolysis has been suggested as having an
acceptable level of distillation energy requirements. M. Galbe
& G. Zacchi, A review of the production of ethanol from
softwood, Applied Microbiology and Biotechnology 59, 618-628
(2002).
[0005] Increasing final product titer may be achieved with higher
solids loadings, but handling high solids introduces mixing
requirements that increase the complexity and energy cost of the
process. Mixing power requirements increase with increasing solids
loadings, so non-conventional mixing techniques have been applied
to biomass reactions at high solids concentration. For example, a
free-fall mixer has been applied to wheat straw loadings of up to
40% solids. Henning Jorgensen, et al., Liquifaction of
lignocelluloses at high solids concentrations, Biotechnology and
Bioengineering 96, 862-870 (2006). Mixing may be accomplished in a
number of other ways, including high consistency mixers, screw
presses, static mixers, or by spray application. One example of
spray application of acid is Yan et al. L. Yan, et al., Dilute
sulfuric acid cycle spray flow-through pretreatment of corn stover
for enhancement of sugar recovery, Bioresource Technology 100,
1803-1808 (2009). High solids may also slow hydrolysis in the
reactor for reasons other than mixing limitations. Jan B.
Kristensen, et al., Yield-determining factors in high solids
enzymatic hydrolysis of lignocellulose, Biotechnology for Biofuels
2, 11 (2009).
[0006] Product titer may also be increased by reducing dilution
steps in hydrolysis and fermentation processes. Methods of
lignocellulose hydrolysis and/or fermentation include separate
hydrolysis and fermentation (SHF--hydrolysis first, then
fermentation) or simultaneous saccharification and fermentation of
lignocelluloses (SSF--hydrolysis and fermentation happens at the
same time). Saccharification and fermentation of lignocellulosic
biomass typically require an acidic and/or a chemical pretreatment
process or an alkaline chemical pretreatment process. In a biomass
pretreatment process without complete biomass hydrolysis, a
pretreated biomass solids stream and a prehydrolysate stream are
generated. The pretreated biomass stream (solids fraction) is rich
in cellulose while the prehydrolysate stream (liquid fraction) is
rich in hemicellulose sugars or hemicellulose oligomers, along with
lignin, extractives, furans, aldehydes, acetic acid, or other
inhibitors that restrict the growth and productivity of a
fermenting organism. Additionally, the prehydrolysate stream
usually has a pH outside of the typical enzymatic hydrolysis pH
range (4.8 to 6.5) or typical fermentation pH range (3.5 to 7.5).
Similarly, the pretreated biomass solids also may also contain
inhibitors and a different pH from the enzymatic hydrolysis pH and
the fermentation pH. Therefore, prehydrolysate and pretreated
biomass conditioning are often needed before an enzymatic
hydrolysis and a fermentation process.
[0007] The prehydrolysate is typically separated from the solids
after pretreatment and conditioned by overliming (Andy Aden,
Biochemical Production of Ethanol from Corn Stover: 2007 State of
Technology Model, NREL/TP-510-43205 (2008)) or ion exchange (J. Y.
Zhu, et al., Ethanol production from SPORL-pretreated lodgepole
pine: preliminary evaluation of mass balance and process energy
efficiency, Applied Microbiology and Biotechnology 86, 1355-1365
(2010)) and fermented separately from the solids fraction. In these
scenarios, it is necessary to add dilution to accomplish pH
changes, enzyme loading, and, in the case of simultaneous
saccharification and fermentation (SSF), fermentation organism
loading.
[0008] The solids fraction of the pretreatment process is typically
washed to remove fermentation inhibitors, then hydrolyzed, and
fermented either separately or together with the prehydrolysate.
Since both the prehydrolysate and the hydrolysate contain sugars
that could be fermented to maximize yield, any water added up to
the point of fermentation reduces the ultimate product titer. This
requires greater energy requirements for separation with
distillation. The high initial viscosity and associated poor mixing
properties of the solids fraction of the pretreated cellulosic
biomass may also reduce the product titer. For lab scale
experimentation, pretreated cellulose is often hydrolyzed at 5%
solids or even lower to enable mixing, and mixing effectiveness
decreases (or requires more power) as solids increase. The
difference between 10% solids and 20% solids going into hydrolysis,
for example, is a factor of two on the ultimate product titer,
resulting in approximately a factor of two in distillation energy
use. G. Zacchi & A. Axelsson, Economic evaluation of
preconcentration in production of ethanol from dilute sugar
solutions, Biotechnology and Bioengineering 34, 223-233 (1989).
Greater hydrolysis reactor volume, fermentor volume and
distillation column volume will be required for lower product titer
liquid handling, potentially tripling the total capital cost of
downstream processing.
[0009] In both SHF and SSF, it has been anticipated that "to
increase the sugar concentration in a future large-scale operation,
it is assumed that the whole slurry after pretreatment would be
used without introducing separation steps that would dilute the
process stream." M. Galbe & G. Zacchi, Applied Microbiology and
Biotechnology 59, 618-628 (2002). Therefore, what is needed is a
method of hydrolyzing and fermenting biomass compositions to
produce product liquids of high titer with minimal dilution steps.
The method would make use of the prehydrolysate to the extent
needed to optimize mixing, enzymatic hydrolysis, and fermentation.
The prehydrolysate stream may be used to accomplish pH changes,
enzyme loading, and, in the case of SSF, fermentation organism
loading to avoid adding dilution water.
SUMMARY
[0010] The present invention provides spray methods of delivering
saccharification enzymes, fermentation organisms, and/or other
hydrolysis or fermentation ingredients onto solid biomass
comprising lignins and/or cellulose. More specifically it relates
to spray methods using a pretreatment prehydrolysate as the
delivery method for a composition to promote hydrolysis or
simultaneous hydrolysis and fermentation processes in solid biomass
to be broken down, thereby reducing the amount of dilution and
increasing the titer of a saccharification or fermentation
product.
[0011] In one non-limiting variation, the present invention
provides a method of producing a saccharification product from a
pretreated biomass composition, the method comprising: a) spraying
a liquid comprising one or more saccharification enzymes onto the
pretreated biomass composition to form an impregnated biomass
composition; and b) maintaining the impregnated biomass composition
under suitable conditions to promote hydrolysis of components in
the impregnated biomass composition to produce the saccharification
product. In some non-limiting variations, the liquid further
comprises one or more fermentation organisms, and wherein the one
or more fermentation organisms ferment sugars produced by
hydrolysis to produce a fermentation product. In some non-limiting
variations, the liquid is a prehydrolysate produced from the
pretreated biomass composition. In other non-limiting variations,
the liquid is a prehydrolysate produced from a second pretreated
biomass composition.
[0012] In another non-limiting variation, the present invention
provides a method of producing a saccharification product from a
pretreated biomass composition comprising a prehydrolysate and
pretreated biomass solids, the method comprising: a) separating the
prehydrolysate from the pretreated biomass solids to form a first
prehydrolysate liquid; b) adding one or more saccharification
enzymes to the first prehydrolysate liquid to form a second
prehydrolysate liquid; c) spraying the second prehydrolysate liquid
onto the pretreated biomass solids to form an impregnated biomass
composition; and d) maintaining the impregnated biomass composition
under suitable conditions to promote enzymatic hydrolysis of the
impregnated biomass to produce the saccharification product. In
some non-limiting variations, the method further comprises: a)
adding one or more fermentation organisms to the saccharification
product; and b) maintaining the saccharification product under
suitable conditions to promote fermentation of sugars produced by
hydrolysis to produce a fermentation product.
[0013] In some non-limiting variations, the pretreated biomass
solids are not rinsed or conditioned before spraying with the
second prehydrolysate. In some non-limiting variations, the methods
disclosed herein further comprise adjusting the pH of the first or
second prehydrolysate to a pH range of 3.0 to 9.0 prior to the
spraying step. In some non-limiting variations, the one or more
saccharification enzymes are selected from the group consisting of
cellulase, beta-glucosidase, xylanase, other hemicellulases, and
mixtures thereof. In some non-limiting variations, the fermentation
organisms are selected from the group consisting of yeast, mold,
algae, bacteria, and mixtures thereof.
[0014] In some non-limiting variations, the methods disclosed
herein further comprise mixing the pretreated biomass composition
with the second prehydrolysate liquid.
[0015] In some non-limiting variations, the pretreated biomass
solids are transported on a screw or a belt conveyer during the
spraying step to form the impregnated biomass composition. In some
variations, the methods disclosed herein further comprise moving
the impregnated biomass composition with a belt or screw conveyer
into a reactor for the hydrolysis.
[0016] In some non-limiting variations, the pretreated biomass
composition comprises wood chips, and the wood chips are size
reduced prior to pretreatment such that prior to size reduction,
50-100% of the wood chips pass through a +7 mm round hole screen,
and after size reduction, 50-100% of the wood chips pass through a
+3 mm round hole screen. In some non-limiting variations, the
pretreated biomass solids are ground up prior to spraying with the
second prehydrolysate liquid.
[0017] In some non-limiting variations, the pretreated biomass
composition is produced from an acid sulfite or acid bisulfite
pretreatment process.
[0018] In some non-limiting variations, the pretreated biomass
composition is produced from an alkaline sulfite or alkaline
bisulfite pretreatment process. In some non-limiting variations,
the pretreated biomass composition is produced from a neutral
chemical pretreatment process or a pretreatment process using water
and no additional chemicals.
[0019] In some non-limiting variations, the fermentation product
has a titer greater than 4.0%. In some non-limiting variations, the
hydrolysis produces a sugar yield of 80-100% of the pretreated
biomass composition. In some non-limiting variations, the
fermentation converts 60-100% of the sugars to the fermentation
product.
[0020] The present invention further provides a method of producing
a fermentation product from a pretreated biomass composition
comprising a prehydrolysate and pretreated biomass solids, the
method comprising: a) separating the prehydrolysate from the
pretreated biomass solids to form a first prehydrolysate liquid; b)
adding one or more saccharification enzymes and one or more
fermentation organisms to the first prehydrolysate liquid to form a
second prehydrolysate liquid; c) spraying the second prehydrolysate
liquid onto the pretreated biomass solids to form an impregnated
biomass composition; and d) maintaining the impregnated biomass
composition under suitable conditions to promote enzymatic
hydrolysis of the impregnated biomass composition and fermentation
of the saccharification product to form the fermentation
product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Smaller, re-chipped chips result in higher sugar
yield compared to larger, screened chips in magnesium bisulfite
pretreatment with 2% sulfuric acid on wood.
[0022] FIG. 2. Batch simultaneous saccharification and fermentation
with Saccharomyces cerevisiae D5a on calcium bisulfite pretreated
softwood and prehydrolysate from re-chipped wood chips.
[0023] FIG. 3. Batch simultaneous saccharification and fermentation
with Saccharomyces cerevisiae T2 on calcium bisulfite pretreated
softwood and prehydrolysate from re-chipped wood chips.
[0024] FIG. 4. Batch simultaneous saccharification and fermentation
with Saccharomyces cerevisiae T2 on calcium bisulfite pretreated
softwood and prehydrolysate from re-chipped wood chips. The
saccharification enzymes and fermentation organisms were added to
the prehydrolysate and spray delivered onto the pretreated wood
chips.
[0025] FIG. 5. Process flow diagram of spraying enzyme(s),
fermenting organism(s), and/or nutrients in a prehydrolysate
diluent onto pretreated biomass solids.
[0026] FIG. 6. Process flow diagram of spraying enzyme(s),
fermenting organism(s), and/or nutrients without a prehydrolysate
diluent onto biomass solids.
[0027] FIG. 7. Process design of spraying enzymes, fermentation
organisms, and/or nutrients onto biomass solids using a conveyor to
transport the solids under a spraying apparatus and into a
hydrolysis and/or fermentation reactor.
DETAILED DESCRIPTION
[0028] The following description sets forth exemplary methods,
parameters and the like. It should be recognized, however, that
such description is not intended as a limitation on the scope of
the present invention but is instead provided as a description of
exemplary embodiments. From these, a person of ordinary skill would
be able to practice the invention without undue
experimentation.
1. DEFINITIONS
[0029] As used herein, "pretreatment" refers to the thermo-chemical
treatment of biomass in order to make cellulose available to
downstream hydrolysis and biologically-based conversion processes.
Unless indicated otherwise, it does not include further processing
steps such as separation of solid and liquid phases of the
pretreatment product or rinsing or conditioning of the solid or
liquid product phases. Similarly, "pretreated biomass solids" refer
to biomass solids that have undergone pretreatment, and unless
otherwise indicated, a pretreated biomass solid has not received
other treatments or processing.
[0030] As used herein, the term "prehydrolysate" refers to the
liquid fraction of the pretreatment reaction mixture.
[0031] As used herein, the term "hydrolysate" refers to the
reaction mixture formed from a prehydrolysate and/or pretreated
biomass solids after having undergone enzymatic hydrolysis to at
least partially break down oligosaccharides into sugars.
[0032] As used herein, "saccharification" refers to the enzymatic
hydrolysis of biomass to monomeric and/or oligomeric sugars.
[0033] As used herein, "saccharification product" refers to the
product generated when a saccharification enzyme catalyzes
hydrolysis of biomass, typically pretreated biomass.
[0034] As used herein, a "saccharification enzyme" refers to an
enzyme that is capable of catalyzing the hydrolysis of biomass to
monomeric and/or oligomeric sugars.
[0035] As used herein, "inhibitor(s)" or "byproduct(s)" refer to
side products other than sugars that may be present in both the
prehydrolysate after pretreatment and the hydrolysate after
enzymatic hydrolysis. Inhibitors, in particular, are byproducts
that are capable of interfering with enzymatic hydrolysis or
fermentation.
[0036] As used here in "comminution" refers to a method where
biomass solids are mechanically resized to small or finer particles
with a varied particle size range or distribution. In some
non-limiting variations, the biomass solids may be woody trunks or
branches resized to small or finer chips with a varied chip size
range or distribution. In some non-limiting variations, 50-100% of
the biomass solids are able to pass through a +7 mm diameter round
hole screen prior to comminution. In other non-limiting variations,
50-100% of the biomass solids are able to pass through a +3 mm
diameter round hole screen after comminution and may be referred to
"re-chipped" wood chips or biomass solids. In some preferred
variations, 50-100% of the "re-chipped" wood chips or biomass
solids are able to pass through a +7 mm diameter round hole screen
and the remaining 0-50% of the "re-chipped" wood chips or biomass
solids are able to pass through a +3 mm diameter round hole screen.
In some preferred variations, approximately 20-25% of the
"re-chipped" wood chips or biomass solids are able to pass through
a +7 mm diameter round hole screen and the remaining 75-80% of the
"re-chipped" wood chips or biomass solids are able to pass through
a +3 mm round hole screen.
[0037] As used herein, "spraying" refers to delivering a
composition, typically a liquid or slurry or suspension, as a
plurality of drops spread relatively evenly onto a material. As
used herein, the term "spraying" also encompasses "sprinkling,"
where a composition is delivered sparsely and with small scale
non-uniformity onto a material. In some non-limiting variations,
the spraying may be accomplished with a spraying nozzle, a
sprinkling nozzle, or some other mechanism such as a flat plate
mounted over a conveyor. In some non-limiting variations, the
spraying may deposit a fine and constant mist of the composition
onto the material or coarse droplets of the composition onto the
material, or a mixture of fine and coarse droplets. In some
non-limiting variations, the composition or liquid being applied
may or may not include solid material, such as biomass particles or
other products generated from biomass pretreatment processes. In
some non-limiting variations, the composition may be pressurized in
a container with compressed gas or air or with a pump or with a
gravity head pressure and may exit the container through a spray
nozzle that sprays the mixture onto the other material.
[0038] As used herein, when the term "about" modifies a number, the
term is defined as "approximately," and the number should be
interpreted to cover a range that includes its recited value and
the experimental error in obtaining the number.
2. DESCRIPTION OF THE INVENTION
[0039] The present invention provides spray methods of delivering
saccharification enzymes, fermentation organisms, and/or other
hydrolysis or fermentation ingredients onto solid biomass including
lignins and/or cellulose. More specifically it relates to spray
methods using a pretreatment prehydrolysate as the delivery method
for a composition to promote hydrolysis or simultaneous hydrolysis
and fermentation processes in solid biomass to be broken down,
thereby reducing the amount of dilution and increasing the titer of
a saccharification or fermentation product.
[0040] In cellulosic ethanol or other bioproduct fermentation, high
solids loading in biomass hydrolysis is one way to achieve high
sugar titer, high product concentration, and high productivity in
order to reduce capital cost, operation cost, and energy
consumption. Hydrolysis or fermentation at an initial 14% or higher
solids loading may encounter initial mass transfer difficulties
with enzyme and/or fermentation organism and nutrients, thus
slowing down the hydrolysis or fermentation process. The methods
disclosed herein involve spraying hydrolysis and/or fermentation
ingredients onto pre-treated biomass solids, e.g., high solids,
fibrous cellulose materials to achieve effective mass transfer and
maintain high solids loading. Spray application may be as effective
for both SHF and SSF as thorough mixing, and increases efficiency
by reducing energy usage and reducing or eliminating the need for
physical mixing of the biomass with the ingredients added to
promote breaking down the solids. The prehydrolysate delivery
method requires no additional water for pretreated biomass washing
and thus minimizes water usage and wastewater treatment, yet its
overall efficiency for converting biomass such as wood chips into
water-soluble sugars and/or ethanol is comparable to conventional
mixed SSF or SHF processes.
[0041] The methods of the present invention provide a method of
producing a saccharification product from a pretreated biomass
composition, the method comprising: a) spraying a liquid including
one or more saccharification enzymes onto the pretreated biomass
composition to form an impregnated biomass composition; and b)
providing operating conditions for enzymatic hydrolysis of the
impregnated biomass composition to produce a saccharification
product that typically includes sugars and/or hemicelluloses that
are relatively water soluble. The spraying liquid may be prepared
by admixing the one or more saccharification enzymes with a
prehydrolysate liquid and may or may not contain suspended
particles or solids. The prehydrolysate liquid is a composition
produced by pretreatment of biomass, and can be the same
prehydrolysate produced in the pretreatment process that produced
the pretreated biomass for use in this method or a different
prehydrolysate produced from a different pretreated biomass. In
some non-limiting variations, the spraying composition further
includes one or more fermentation organisms, and the one or more
fermentation organisms ferment the saccharification products as
they are formed to produce a fermentation product. In these
non-limiting variations, the spraying liquid includes both the
saccharification enzyme(s) and fermentation organism(s), and the
enzymatic hydrolyzing and microbial fermenting may be carried out
in one reactor. The spraying composition may further include acid,
base or buffer to adjust pH to a suitable range; nutrients to
sustain growth of a fermentation microorganism; and/or substances
to support or enhance the function of enzymes present to help break
down cellulose, hemicelluloses or other components of the
pretreated biomass.
[0042] The methods of the present invention further provide a
method of producing a saccharification product from a pretreated
biomass composition including a prehydrolysate and pretreated
biomass solids, wherein the method includes: a) separating the
prehydrolysate from the pretreated biomass solids to form a first
prehydrolysate liquid; b) adding one or more saccharification
enzymes to the first prehydrolysate liquid to form a second
prehydrolysate liquid; c) spraying the second prehydrolysate liquid
onto the pretreated biomass solids to form an impregnated biomass
composition, preferably without rinsing or treating the pretreated
biomass solids; and d) maintaining the impregnated biomass
composition under suitable conditions to promote enzymatic
hydrolysis of the impregnated biomass composition to produce the
saccharification product. In one non-limiting variation, the method
further includes e) adding one or more fermentation organisms to
the saccharification product; and f) maintaining the
saccharification product under suitable conditions to promote
fermentation of sugars produced by hydrolysis to produce a
fermentation product. In another non-limiting variation, one or
more fermentation organisms are added to the first prehydrolysate
liquid in addition to the one or more saccharification enzymes to
form the second prehydrolysate liquid, the second prehydrolysate
liquid including both the saccharification and fermentation
organisms is sprayed onto the pretreated biomass solids, and the
one or more fermentation organisms ferment the sugars produced by
saccharification to produce a fermentation product, typically as a
liquid also. In these non-limiting variations, the enzymatic
hydrolyzing and fermenting may be carried out in one reactor and
may occur concurrently, or they may be done sequentially by
performing the saccharification process and then adding
fermentation organisms and any needed additives to promote
fermenting of the sugars from the saccharification.
[0043] In some non-limiting variations, the methods further include
adjusting the pH of the first or second prehydrolysate liquid to a
pH range of 3.0 to 9.0 prior to the spraying step.
[0044] In some non-limiting variations, the one or more
saccharification enzymes include cellulase, beta-glucosidase,
xylanase, other hemicellulases, and mixtures thereof. In some
non-limiting variations, the fermentation organisms include yeast,
mold, algae, bacteria, and mixtures thereof.
[0045] In some non-limiting variations, the methods of the
invention further include mixing the pretreated biomass composition
with the second prehydrolysate composition, typically by spraying
or sprinkling the second prehydrolysate liquid onto the pretreated
biomass.
[0046] In some non-limiting variations, the pretreated biomass
solids are transported on a screw conveyer or a belt conveyer
during the spraying step to form the impregnated biomass
composition. In some non-limiting variations, the methods of the
invention further include moving the impregnated biomass
composition with a screw or belt conveyer into a reactor for the
enzymatically hydrolyzing or the enzymatically hydrolyzing and
fermenting steps.
[0047] In some non-limiting variations, the methods of the
invention further include circulating the impregnated biomass
composition inside the reactor. In some preferred variations, a
slurry pump, a slow agitator, or a slow impeller circulates the
impregnated biomass composition.
[0048] In some non-limiting variations, the pretreated biomass
composition includes wood chips. In some preferred variations, the
wood chips are size reduced prior to pretreatment. In some
non-limiting variations, 50-100% of the biomass solids are able to
pass through a +7 mm diameter round hole screen prior to
comminution and are referred to as "screened" wood chips throughout
the disclosure. In other non-limiting variations, 50-100% of the
biomass solids are able to pass through a +3 mm diameter round hole
screen after comminution and are referred to as "re-chipped" wood
chips or "re-chipped" biomass solids throughout the disclosure. In
some preferred variations, 50-100% of the "re-chipped" wood chips
or biomass solids are able to pass through a +7 mm diameter round
hole screen, and the remaining 0-50% of the "re-chipped" wood chips
or biomass solids are able to pass through a +3 mm diameter round
hole screen. In some preferred variations, approximately 20-25% of
the "re-chipped" wood chips or biomass solids are able to pass
through a +7 mm diameter round hole screen, and the remaining
75-80% of the "re-chipped" wood chips or biomass solids are able to
pass through a +3 mm round hole screen.
[0049] In some non-limiting variations, the pretreated biomass
composition is produced from an acid sulfite or acid bisulfite
pretreatment process. In some preferred variations, the pretreated
biomass composition is produced from an alkaline sulfite or
alkaline bisulfite pretreatment process. Pretreatments can use
alkali metal salts or alkaline earth metal salts of bisulfites or
sulfites. In some preferred variations, the pretreated biomass
composition is produced from a neutral chemical pretreatment
process. Pretreatment reactions were conducted for suitable time
periods under suitable conditions to promote the desired process.
For example, in some preferred variations, the pretreated biomass
composition is produced from a pretreatment process using water and
no additional chemicals. In some non-limiting variations, the
pretreatment temperature is ramped from a lower temperature of
about 50-150.degree. C. to a higher temperature of about
150-250.degree. C. over a first period of time and held at the
higher temperature for a second period of time. In some
non-limiting variations the lower temperature is about 75.degree.
C.-125.degree. C. and the higher temperature is about
170-200.degree. C. In some non-limiting variations the lower
temperature is about 80-100.degree. C. and the higher temperature
is about 170-190.degree. C. In some non-limiting variations, the
first period of time is 5-30 minutes and the second period of time
is 10-60 minutes. In some non-limiting variations, the first period
of time is 10-20 minutes and the second period of time is 15-25
minutes.
[0050] Saccharification reactions were conducted for suitable time
periods under suitable conditions to promote the desired process.
For example, in some non-limiting variations, the saccharification
is carried out at 1-20% solids loading (by weight). In other
non-limiting variations, the saccharification is carried out at
2-10% solids loading. In other non-limiting variations, the
saccharification is carried out at about 5% solids loading. In some
non-limiting variations, the pH of the saccharification reaction is
about 2-7. In some non-limiting variations, the pH is about 3-6. In
other non-limiting variations, the pH is about 4-5. In some
non-limiting variations, the saccharification conditions are
maintained for 24-72 hours. In some non-limiting variations, the
saccharification conditions are maintained for 36-60 hours. In some
non-limiting variations, the saccharification conditions are
maintained for about 48 hours. In some non-limiting variations,
cellulase, beta-glucosidase, xylanase, other hemicellulases, or
mixtures of any combination of enzymes are used in the hydrolysis
reactions. In some non-limiting variations 5-25 FPU cellulase is
used in the enzymatic hydrolysis. In some non-limiting variations,
10-20 FPU cellulase is used in the enzymatic hydrolysis. In some
non-limiting variations, about 16 FPU cellulase is used in the
enzymatic hydrolysis. In some non-limiting variations, 10-30 U of
beta-glucosidase is used in the enzymatic hydrolysis. In some
non-limiting variations, 15-25 U of beta-glucosidase is used in the
enzymatic hydrolysis. In some non-limiting variations, about 20 U
of beta-glucosidase is used in the enzymatic hydrolysis. In some
non-limiting variations, 5-20 U of xylanase is used in the
enzymatic hydrolysis. In some non-limiting variations, 10-15 U of
xylanase is used in the enzymatic hydrolysis. In some non-limiting
variations, about 9-10 U of xylanase is used in the enzymatic
hydrolysis. In some non-limiting variations, the saccharification
produces a sugar yield of 80-100% of the pretreated biomass
composition.
[0051] Fermentation reactions were conducted for suitable time
periods under suitable conditions to promote the desired process.
In some non-limiting variations, saccharification may be carried
out first, followed by fermentation (SHF). In other non-limiting
variations, saccharification and fermentation reactions may be
carried out in the same reactor, by adding both saccharification
enzymes and fermentation organisms to the pretreated biomass
composition in the reactor, and maintaining the reactor under
suitable conditions that promote both saccharification and
fermentation (SSF). In some non-limiting variations, the
concentration of the fermentation organism is 0.5-5 g/L. In other
non-limiting variations, the concentration of the fermentation
organism is 1-3 g/L. In other non-limiting variations, the
concentration of the fermentation organism is about 2 g/L. In some
non-limiting variations, the pH of the fermentation reaction is
about 4-7. In some non-limiting variations, the pH of the
fermentation reaction is about 6-7. In some non-limiting
variations, buffers and nutrients may be added to the fermentation
reactions, such as, for example, sodium citrate, peptone, or corn
steep liquor.
[0052] In some non-limiting variations, the fermentation or SSF
conditions are maintained for 24-72 hours. In some non-limiting
variations, the fermentation or SSF conditions are maintained for
36-60 hours. In some non-limiting variations, the fermentation or
SSF conditions are maintained for about 48 hours. In some
non-limiting variations, the fermentation product has a titer
greater than 4%. In some non-limiting variations, the fermentation
product has a titer of 4-20%. In other non-limiting variations, the
fermentation product has a titer of 4-10%. In other non-limiting
variations, the fermentation product has a titer of 4-6%. In some
non-limiting variations, the fermentation converts 60-100% of
sugars to the fermentation product, based on the type of sugars the
fermenting organisms are selected to process. In some non-limiting
variations, the fermentation converts 80-90% of sugars to the
fermentation product.
EXAMPLES
[0053] Reagents
[0054] Magnesium bisulfite (Sigma Catalog #398233) was purchased as
a 30% w/w solution. A calcium bisulfite solution of 4 to 7% was
made by purging a sulfur dioxide stream into a calcium oxide
solution or slurry in the laboratory. Sulfuric acid (95.8% assay
and Catalog #A-300-212) was purchased from Fisher scientific.
Sodium Citrate (Sigma Catalog #71498), potassium hydroxide (Sigma
Catalog #P5958), and urea (Sigma Catalog #U5128) were purchased
from Sigma-Aldrich. These reagents were diluted with deionized
water or the prehydrolysate to the appropriate concentration for
loading the pre-calculated amount into the reactor. All the
reported bisulfite reagents and acid loadings are based on the oven
dry (OD) weight of the biomass.
[0055] Cellulase (Celluclast, Sigma Catalog #C-2730);
beta-glucosidase (Novozymes-188, Sigma Catalog #C-6105); and
xylanase (Novozymes NS50030; 500 FXU/g) were used in the enzymatic
hydrolysis experiments. The activity of celluclast was found to be
approximately 80 FPU/ml as measured in Weyerhaeuser Lab (Seattle,
Wash.) and activity of beta-glucosidase was reported to be 250
CBU/g (300 CBU/ml) by the supplier (Novozymes). These enzymes were
stored in refrigerator below 4.degree. C.
[0056] The yeast strain Saccharomyces cerevisiae D5a (ATCC 200062)
was obtained from American Type Culture Collection (ATCC, Manassas,
Va.) and was used in Example 4. Another yeast strain Saccharomyces
cerevisiae T2 was obtained from Dr. Sheldon Duff at the University
of British Columbia and was used in Example 5.
Example 1
Reduction of Chip Size Prior to Calcium Bisulfite Pretreatment
[0057] For woody feedstocks in particular, reducing inhibitors
byproducts in a pretreatment process may be achieved by reducing
the size of the wood chip to enable less severity of time or
temperature. Because a small quantity of inhibitor byproducts is
present in the prehydrolysate liquid, the prehydrolysate may not
require a separate conditioning process to remove the inhibitors
prior to using the prehydrolysate as a diluent for enzymatic
hydrolysis.
[0058] Wood pretreatment and hydrolysis may be carried out on
finely ground wood. Hemicellulose removal during the pretreatment
process is known to reduce energy requirements for particle
comminution; therefore, comminution of wood particles after
pretreatment may be more energy efficient. However, comminution
prior to pretreatment may also improve the pretreatment
process.
[0059] Commercial pine wood chips were obtained from a pulp mill in
the southern U.S. These pine wood chips represent the "screened"
chips in Table 1 and are representative of the feed to a pulp
digester in a commercial pulp and paper operation. A sub-sample of
these chips was fractured with a BearCat garden chipper with a
3/4'' screen to obtain the "re-chipped" fiber distribution shown
below in Table 1. For the re-chipped chips, the 3 mm round hole
fines were removed to avoid circulation problems in the lab
pretreatment reactor.
TABLE-US-00001 TABLE 1 Size classifications of pretreated and
hydrolyzed chip size distributions. Size Classification (Percent of
Total) Chip +8 mm +7 mm +3 mm -3 mm Treatment thickness round hole
round hole round hole Screened 5.5% 85.8% 6.8% 0.5% Re-chipped 0.0%
23.5% 76.5% 0.0%
[0060] The chip size distributions shown in Table 1 were pretreated
in separate runs of a one cubic foot reactor with a sulfite
pretreatment consisting of 12% calcium bisulfite on wood with a
two-step temperature schedule: ramp from 90.degree. C. to
160.degree. C. in 15 minutes, hold at 160.degree. C. for 30
minutes, ramp to 180.degree. C. quickly (6 minutes), and hold at
180.degree. C. for 20 minutes. This temperature scheme was
determined to be optimal for reducing pretreatment severity and
inhibitor production under calcium bisulfite pretreatment
Conditions. The solubilization of wood in each case was identical
at approximately 23% on dry wood basis. The prehydrolysate was
collected, and the pretreated, unwashed solids were ground in an
Alpine grinder. The prehydrolysate was then recombined with the
ground solids and sodium citrate was added as a buffer at a 50 mmol
concentration. The composition was subjected to enzymatic
hydrolysis at 5% solids with 16 FPU cellulase/dry gram of
pretreated solids and 20 U beta-glucosidase/dry gram of pretreated
solids at pH 4.8. After 48 hours, the quantity of hydrolyzed sugars
was higher and the quantity of inhibitors was lower for the smaller
re-chipped size distribution, as shown in Table 2. In addition, the
re-chipped has a higher hydrolysis conversion of sugars than the
screened: 88% (re-chipped) and 79% (screened). The total furan
inhibitors in the prehydrolysate liquor were also lower for the
re-chipped over the screened: 0.61% (re-chipped) and 1.02%
(screened).
TABLE-US-00002 TABLE 2 Effect of particle size reduction on
prehydrolysate composition after pretreatment and on total sugar
conversion after 48 hour enzymatic hydrolysis. Un- Re-Chipped
Screened pretreated (smaller chip (larger Wood size) chip size)
Prehydrolysate Composition (% as polymer on wood) Glucan 39.5%
1.30% 1.30% Xylan + Galactan + Mannan 18.67% 6.36% 5.70% Arabinan
1.01% 0.25% 0.25% Total Sugars (as polymers) 59.18% 7.92% 7.25% HMF
in Liquor 0.24% 0.30% Furfural in Liquor 0.37% 0.72% Total Furans
0.61% 1.02% Total Sugar Conversion (Prehydrolysate + Hydrolysate, %
of theoretical except for wood) Glucan 100.5% 89.7% Xylan +
Galactan + Mannan 63.2% 58.0% Arabinan 70.3% 69.9% Total Sugars (as
polymers) 59.18% 88.2% 79.3% on wood
Example 2
Reduction of Chip Size Prior to Magnesium Bisulfite
Pretreatment
[0061] Pretreatment tests with magnesium bisulfite 9.6% (wt % on
dry wood basis) and sulfuric acid 2% (wt % on dry wood basis) on
re-chipped chips and larger chips were compared for the sugar
hydrolysis yields and furan formation. Two different pretreatment
temperature schemes were tested: (1) a controlled ramp from
90.degree. C. to 180.degree. C. in 15 minutes and held at
180.degree. C. for 20 minutes; and (2) a slower, controlled from
90.degree. C. to 180.degree. C. in 60 minutes and held at
180.degree. C. for 20 minutes. After the pretreatment, the
pretreated and unwashed solids were ground in an Alpine grinder,
and enzymatic hydrolysis was conducted in 5% pretreated materials
using the same procedure as in Example 1 above. The sugar
conversion yields are compared in FIG. 1.
[0062] The magnesium bisulfite pretreatment runs produced similar
results as was observed for calcium bisulfite in Example 1. Under
both temperature schemes, the smaller chips had better hydrolysis
characteristics in the one cubic foot reactor. The smaller chip
size distribution had a 4% to 8% improvement in total sugar yield
upon hydrolysis (FIG. 1). However, the magnesium bisulfite/sulfuric
acid runs produced furan concentrations greater than 1% on wood
compared to the 0.61% on wood with calcium bisulfite with no
exogenous sulfuric acid added (See FIG. 1 and Table 2, smaller chip
size). The calcium bisulfite conditions are preferred over the
magnesium bisulfite conditions due to lower inhibitor formation and
the ability to use the bisulfite reagent without additional acid.
Also, the re-chipped chips are preferred over the screened chips
due to higher yields of enzymatic hydrolysis. The slower
temperature scheme on re-chipped chips significantly reduced furan
formation in the cook liquor while the faster temperature ramp
scheme showed insignificant impact on furan formation.
Example 3
Spray Application of Enzymes in Prehydrolysate for High Solids
Enzymatic Hydrolysis
[0063] High solids enzymatic hydrolysis was tested on calcium
bisulfite pretreated re-chipped chip biomass, using the
prehydrolysate as an enzyme delivery vehicle in a spray
application. In one single spray application, the enzyme(s) are
delivered to the pretreated biomass, the pretreated biomass is
neutralized, and mixing of the pretreated biomass and enzymes are
achieved. Washing the pretreated biomass with water prior to
hydrolysis was not needed. Mechanical agitation and high viscosity
mixing were unnecessary for the pretreated biomass hydrolysis.
[0064] The re-chipped chips were first pretreated with calcium
bisulfite (12 wt % on wood) in the one cubic foot digester in a
two-step temperature scheme: ramp from 90.degree. C. to 160.degree.
C. in 15 minutes, hold at 160.degree. C. for 30 minutes, ramp from
160.degree. C. to 180.degree. C. in 9.5 minutes, hold at
180.degree. C. for 20 minutes. The prehydrolysate was collected and
the pretreated, unwashed solids were ground in an Alpine grinder.
The prehydrolysate and the ground pulp material had an acidic pH of
1.46.
[0065] The prehydrolysate was first adjusted to pH 7.5 with a base,
such as potassium hydroxide. Sodium citrate was added to the
prehydrolysate at a 50 mmol concentration. Then, the prehydrolysate
was sterilized in an autoclave at 121.degree. C. for 20 minutes. If
the pH changed after sodium citrate addition and sterilization, the
pH was readjusted back to pH 7.5. To the pH 7.5 prehydrolysate,
enzymes were added at 16 FPU cellulase/dry gram of pretreated
solids, 20 U beta-glucosidase/dry gram of pretreated solids and 9 U
xylanase/dry gram of pretreated solids. The prehydrolysate mixture
containing the enzymes was transferred into a container with a
spray nozzle.
[0066] The ground, pretreated pulp was sterilized in an autoclave
at 121.degree. C. for 20 minutes. The sterile pulp was laid on a
strip of sterile aluminum foil 0.5 ft wide.times.1.0 ft long to
simulate a small conveyor belt session. The pulp layer was evenly
sprayed by the enzyme mixture prepared above in 100%
prehydrolysate. The sprayed pulp was then transferred into a flask
and incubated at 50.degree. C. for enzymatic hydrolysis without any
agitation. Two sprayed hydrolysis tests were carried out, one at
15% and the other at 20% pulp loading. After the spraying process,
the sprayed pulp showed a pH of .about.5.5 for the 15% pulp test
and a pH of .about.5.0 for the 20% pulp test. The test matrix is
shown in Table 3, below.
TABLE-US-00003 TABLE 3 Spray hydrolysis matrix. Test Pulp Load
Prehydrolysate + No. (Dry wt %) Wet Pulp (g) Enzyme (ml) 1 15 35.2
64.8 2 20 46.9 53.1
[0067] Within 24 hours, both tests showed complete liquefication of
the biomass solids, and the total sugar titer and yield results are
showed in Table 4. The yield was calculated in glucan conversion to
glucose in the pretreated pulp materials. In both test cases at 48
hours, glucose yield was achieved at 97.0% and 87.0%, respectively
for the 15% and 20% pulp loading.
TABLE-US-00004 TABLE 4 Spray hydrolysis yields on glucan. Test Pulp
Load (Dry Glucose Yield Glucose Yield00 No. wt %) (%) at 24 hr (%)
at 48 hr 1 15 82.9 97.0 2 20 80.0 87.0
Example 4
High Solids, Simultaneous Saccharification and Fermentation with
Saccharomyces cerevisiae D5a (ATCC 200062)
[0068] High solids simultaneous saccharification and fermentation
was tested on calcium bisulfite pretreated re-chipped chip biomass
to demonstrate the effectiveness of the low level of inhibitors
obtained. The calcium bisulfite pretreated softwood was ground in
an Alpine grinder without any refining water or prehydrolysate as
diluent. The calcium bisulfite pretreated softwood was also used
without any water washes. The refined pulp was subsequently
fermented using a simultaneous saccharification fermentation (SSF)
method.
[0069] The frozen yeast stock (Saccharomyces cerevisiae D5a (ATCC
200062)) was first grown in a complex medium containing yeast
extract 1% and peptone 2%, supplemented with 3% glucose. The stock
seed culture was incubated in a shake flask on an orbital shaking
incubator controlled at 38.degree. C. and 200 rpm. The yeast seed
culture was then centrifuged, and the yeast seed pellet was
dissolved in a small volume of 100 mmol sodium citrate buffer. The
yeast seed was inoculated at 2 g/L dry cell weight in the
fermentation tests.
[0070] The fermentation was conducted in 50 ml volume in 125 ml
Erlenmeyer shake flasks. Corn steep liquor was added at 4% as
nutrients to the fermentation medium, supplemented with 0.11% urea.
Pretreated pulp and prehydrolysate were sterilized separately in an
autoclave at 121.degree. C. for 20 minutes before fermentation. The
SSF was initiated by combining the pretreated pulp (13.6%),
prehydrolysate (undiluted), yeast seed (2 g/L), corn steep liquor
(4%), cellulase (16 FPU/g pulp), beta-glucosidase (20 units/g
pulp), and xylanase (9 U/g pulp). After all the ingredients were
combined, the pulp materials were mixed using a stainless steel
spatula, or a pipette, until the pulp materials and other materials
were combined into a homogeneous, wet slurry. The pulp materials
and the prehydrolysate occupied 80.4% of the total fermentation
volume, and the remaining 19.6% volume included the enzymes,
nutrients, and yeast seed. Fermentation in shake flasks was
controlled at 38.degree. C. and 150 rpm. Fermentation pH was
controlled at .about.pH 6.5 with a 50 mmol sodium citrate buffer
(solid sodium citrate was added and pH was adjusted) by periodic pH
testing and readjustment. The fermentation data is shown in FIG. 2.
The ethanol production was complete in 48 hours, and an ethanol
concentration of 4.4% was achieved. This represents 65.1% of the
total sugars being converted to ethanol, or 66.6 gallons/standard
ton of wood (or 1816 lbs of wood). The yeast used does not ferment
xylose or arabinose, so the ethanol yield would be higher with a
pentose-fermenting organism.
Example 5
High Solids, Simultaneous Saccharification and Fermentation with
Saccharomyces cerevisiae T2
[0071] The Saccharomyces cerevisiae T2 was used in fermentation of
calcium bisulfate pretreated re-chipped chips. The yeast seed was
first grown in a complex medium containing yeast extract 1% and
peptone 2%, supplemented with 3% glucose. The stock seed culture
was incubated in a shake flask on an orbital shaking incubator
controlled at 38.degree. C. and 200 rpm. The yeast seed culture was
then centrifuged and the yeast seed pellet was dissolved in a small
volume of 100 mmol sodium citrate buffer. The yeast seed was
inoculated at 2 g/L dry cell weight in the fermentation tests.
[0072] The fermentation was conducted in 50 ml volume in 125 ml
Erlenmeyer shake flasks. Corn steep liquor was added at 4% as
nutrients to the fermentation medium, supplemented with 0.11% urea.
Pretreated pulp and prehydrolysate were sterilized separately in an
autoclave at 121.degree. C. for 20 minutes before fermentation. The
SSF was initiated by adding the pretreated pulp (14.0%), liquor
(full strength), yeast seed (2 g/L), corn steep liquor (4%),
cellulase (16 FPU/g pulp), beta-glucosidase (20 units/g pulp), and
xylanase (9 U/g pulp). After all the ingredients were added, the
initial mixing was completed by a vigorous mixing using a stainless
steel spatula, or a pipette, until all the pulp materials showed a
homogeneous wet slurry. The total pulp materials and the
prehydrolysate occupied 80.7% of the total fermentation volume and
the rest 19.3% volume was from enzymes, nutrients, and yeast seed.
Fermentation in shake flasks was controlled at 38.degree. C. and
150 rpm. Fermentation pH was controlled at .about.pH 6.5 by a 50
mmol sodium citrate buffer (solid sodium citrate was added and pH
was adjusted) and by periodic pH checking and readjustment. The
fermentation data is shown in FIG. 3. The ethanol production was
complete in 48 hours, and an ethanol concentration of 4.4% was
achieved. This represents 64.0% of the total sugars being converted
to ethanol, or 65.3 gallons/standard ton of wood.
Example 6
Spray Application of Enzymes and Organisms in Prehydrolysate for
High Solids Simultaneous Saccharification and Fermentation
[0073] High solids simultaneous saccharification and fermentation
was tested on calcium bisulfite pretreated re-chipped chip biomass
using a high efficient enzyme and fermenting organism delivery
method. In this method, the enzyme(s) and the fermenting organism
delivery to pretreated biomass, the neutralization of the
pretreated biomass, and the mixing were completed in one single
spraying step. The pretreated biomass washing with water was not
needed. Mechanical agitation for high solids and high viscosity
mixing was unnecessary in the beginning of the high solids
simultaneous saccharification and fermentation before its
liquefaction.
[0074] The pretreated pulp materials were the same as in Example 3.
The prehydrolysate was first adjusted to pH 7.5 with a base, such
as potassium hydroxide. Sodium citrate was added to the
prehydrolysate at a 50 mmol concentration. The prehydrolysate was
sterilized in an autoclave at 121.degree. C. for 20 minutes. The pH
was readjusted back to pH 7.5 if there was a change. To the pH 7.5
prehydrolysate, SSF ingredients were added, including
enzymes--cellulase (16 FPU/g pulp), beta-glucosidase (20 U/g pulp),
xylanase (9 U/g pulp), corn steep liquor (4%), urea (0.11%), and
yeast seed Saccharomyces cerevisiae T2 (2 g/L). The mixture of
enzymes, yeast seed, nutrients and prehydrolysate was transferred
into a container with a spray nozzle, to be used in the following
sprayed SSF test.
[0075] The ground pretreated-pulp was sterilized in an autoclave at
121.degree. C. for 20 minutes. The sterile pulp was laid on a strip
of sterile aluminum foil 0.5 ft wide.times.1.0 ft long to simulate
a small conveyor belt session. The pulp layer was evenly sprayed by
the mixture of enzymes, yeast seed and nutrients prepared 100%
prehydrolysate. The sprayed pulp was then transferred into a bottle
and incubated at 38.degree. C. for (SSF) ethanol fermentation
without any agitation.
[0076] Three test sets were set up: one set for sprayed SSF, one
set for mixed SSF and one set for top-addition non-well mixed SSF.
The sprayed SSF was described above. After ingredients were added,
the pre-mixed SSF was set up by a vigorous mixing using a stainless
steel spatula or a pipette until all the pulp materials showed a
homogeneous wet slurry. The top-addition non-well mixed SSF was set
up by adding the pH 7.5 prehydrolysate, enzymes, yeast seed and
nutrients to the pulp in the fermentation bottle without further
mixing.
[0077] Ethanol titer and yield results are shown in the following
Table 5. The sprayed SSF had almost identical ethanol titers and
yields on the mixture of pretreated pulp and prehydrolysate as the
pre-mixing SSF, indicating that the spraying delivery of enzymes,
yeast seed and nutrients prepared in 100% prehydrolysate was
effective. One single step of spraying could accomplish mixing as
well as pulp neutralization to the appropriate simultaneous
enzymatic hydrolysis and fermentation pH or SSF pH (FIG. 4). This
was accomplished without a mechanical mixing step by an impeller.
The control non-mixing SSF showed poor ethanol fermentation titer
and yields.
TABLE-US-00005 TABLE 5 Ethanol yields of simultaneous
saccharification and fermentation yields on 14.9% pulp using spray
delivery or pre-mixing of the enzymes and organisms. Test Mixing
Max Ethanol Ethanol Yield Ethanol Yield No. Method Titer (%) (%) on
Sugar (gal/standard ton) 1 Spray 4.79 65.0 66.4 2 Spray 4.83 65.5
66.9 3 Pre-mixing 4.83 65.5 66.9 4 Pre-mixing 4.88 66.2 67.6 5 No
mixing 0.92 12.5 12.7 6 No mixing 1.01 13.7 14.0
Example 7
Process Flow Diagram of Spraying Enzyme(s), Fermenting Organism(s),
and Nutrients in a Prehydrolysate Diluent onto Pretreated Biomass
Solids
[0078] The process flow diagram shown in FIG. 5 summarizes the
integration of an effective spraying delivery of fermenting
organism and/or enzymes and nutrients dissolved in a prehydrolysate
diluent to an acidic or basic pretreated pulp material to achieve a
proper final pH for either enzymatic hydrolysis and/or simultaneous
saccharification fermentation.
Example 8
Process Flow Diagram of Spraying Enzyme(s), Fermenting Organism(s),
and Nutrients without a Prehydrolysate Diluent onto Biomass
Solids
[0079] The process flow diagram shown in FIG. 6 summarizes the
integration of an effective spraying delivery of fermenting
organism and/or enzymes and nutrients without a prehydrolysate
diluent to an acidic or basic pretreated pulp material to achieve a
proper final pH for either enzymatic hydrolysis and/or simultaneous
saccharification fermentation.
Example 9
Process Design for Spraying Enzymes, Fermentation Organisms, and/or
Nutrients onto Biomass Solids Using a Conveyor to Transport the
Biomass Solids Under the Spraying Apparatus and into a Reactor
[0080] This process design shown in FIG. 7 utilizes spraying to
deliver a mixture of prehydrolysate, fermenting organism(s) and/or
enzyme(s) is simple in structure, less in capital cost, low in
maintenance, and effective to evenly distribute enzymes, nutrients,
and fermenting cells to pretreated fibers or pulp materials.
Enzymatic hydrolysis ingredients or fermentation ingredients can be
delivered in one single step to accomplish both mixing and
pretreated material neutralization.
[0081] Although the methods described herein have been described in
connection with some variations, it is not intended to be limited
to the specific form set forth herein. Rather, the scope of the
methods described herein is limited only by the claims.
Additionally, although a feature may appear to be described in
connection with particular variations, one skilled in the art would
recognize that various features of the described variations may be
combined in accordance with the methods described herein.
ENUMERATED EMBODIMENTS
[0082] The following enumerated embodiments are representative of
some aspects of the invention.
[0083] 1. A method of producing a saccharification product from a
pretreated biomass composition, the method comprising:
[0084] a) spraying a liquid comprising one or more saccharification
enzymes onto the pretreated biomass composition to form an
impregnated biomass composition; and
[0085] b) maintaining the impregnated biomass composition under
suitable conditions to promote hydrolysis of components in the
impregnated biomass composition to produce the saccharification
product.
[0086] 2. The method of embodiment 1, wherein the liquid further
comprises one or more fermentation organisms, and wherein the one
or more fermentation organisms ferment sugars produced by
hydrolysis to produce a fermentation product.
[0087] 3. A method of producing a saccharification product from a
pretreated biomass composition comprising a prehydrolysate and
pretreated biomass solids, the method comprising:
[0088] a) separating the prehydrolysate from the pretreated biomass
solids to form a first prehydrolysate liquid;
[0089] b) adding one or more saccharification enzymes to the first
prehydrolysate liquid to form a second prehydrolysate liquid;
[0090] c) spraying the second prehydrolysate liquid onto the
pretreated biomass solids to form an impregnated biomass
composition; and
[0091] d) maintaining the impregnated biomass composition under
suitable conditions to promote enzymatic hydrolysis of the
impregnated biomass composition to produce the saccharification
product.
[0092] 4. The method of embodiment 3, wherein the method further
comprises:
[0093] a) adding one or more fermentation organisms to the
saccharification product; and
[0094] b) maintaining the saccharification product under suitable
conditions to promote fermentation of sugars produced by hydrolysis
to produce a fermentation product.
[0095] 5. The method of either of embodiments 3 or 4, wherein the
pretreated biomass solids are not rinsed or conditioned before
spraying with the second prehydrolysate.
[0096] 6. The method of any of embodiments 3-5, further comprising
adjusting the pH of the first or second prehydrolysate liquid to a
pH range of 3.0 to 9.0 prior to the spraying step.
[0097] 7. The method of any of embodiments 1-6, wherein the one or
more saccharification enzymes are selected from the group
consisting of cellulase, beta-glucosidase, xylanase, other
hemicellulases, and mixtures thereof.
[0098] 8. The method of any of embodiments 2, 4-7, wherein the
fermentation organisms are selected from the group consisting of
yeast, mold, algae, bacteria, and mixtures thereof.
[0099] 9. The method of any of embodiments 3-8, further comprising
mixing the pretreated biomass composition with the second
prehydrolysate liquid.
[0100] 10. The method of any of embodiments 1-9, wherein the
pretreated biomass solids are transported on a screw or a belt
conveyer during the spraying step to form the impregnated biomass
composition.
[0101] 11. The method of any of embodiments 1-10, further
comprising moving the impregnated biomass composition with a belt
or screw conveyer into a reactor prior to hydrolysis, fermentation,
or hydrolysis and fermentation.
[0102] 12. The method of embodiment 11, further comprising
circulating the impregnated biomass composition inside the
reactor.
[0103] 13. The method of embodiment 12, wherein a slurry pump, a
slow agitator, or a slow impeller circulates the impregnated
biomass composition.
[0104] 14. The method of any of embodiments 3-13, wherein the
pretreated biomass composition comprises wood chips, and wherein
the wood chips are size reduced prior to pretreatment, such that
prior to size reduction, 50-100% of the wood chips pass through a
+7 mm round hole screen, and after size reduction, 50-100% of the
wood chips pass through a +3 mm round hole screen.
[0105] 15. The method of any of embodiments 3-14, wherein the
pretreated biomass solids are ground up prior to spraying with the
second prehydrolysate liquid.
[0106] 16. The method of any of embodiments 3-15, wherein the
pretreated biomass composition is produced from an acid sulfite or
acid bisulfite pretreatment process.
[0107] 17. The method of any of embodiments 3-15, wherein the
pretreated biomass composition is produced from an alkaline sulfite
or alkaline bisulfite pretreatment process.
[0108] 18. The method of any of embodiments 3-15, wherein the
pretreated biomass composition is produced from a neutral chemical
pretreatment process or a pretreatment process using water and no
additional chemicals.
[0109] 19. The method of any of embodiments 3-18, wherein the
fermentation product has a titer greater than 4.0%.
[0110] 20. The method of any of embodiments 3-19, wherein the
hydrolysis produces a sugar yield of 80-100% of the pretreated
biomass composition.
[0111] 21. The method of any of embodiments 3-20, wherein the
fermentation converts 60-100% of sugars to the fermentation
product.
[0112] 22. A method of producing a fermentation product from a
pretreated biomass composition comprising a prehydrolysate and
pretreated biomass solids, the method comprising:
[0113] a) separating the prehydrolysate from the pretreated biomass
solids to form a first prehydrolysate liquid;
[0114] b) adding one or more saccharification enzymes and one or
more fermentation organisms to the first prehydrolysate liquid to
form a second prehydrolysate liquid;
[0115] c) spraying the second prehydrolysate liquid onto the
pretreated biomass solids to form an impregnated biomass
composition; and
[0116] d) maintaining the impregnated biomass composition under
suitable conditions to promote enzymatic hydrolysis of the
impregnated biomass composition and fermentation of the
saccharification product to form the fermentation product.
[0117] 23. The method of either embodiment 1 or 2, wherein the
liquid is a prehydrolysate produced from the pretreated biomass
composition.
[0118] 24. The method of either embodiment 1 or 2, wherein the
liquid is a prehydrolysate produced from a second pretreated
biomass composition.
[0119] Although individual features of the methods described herein
may be included in different claims, these may be advantageously
combined, and the inclusion in different claims does not imply that
a combination of features is not feasible and/or advantageous.
Also, the inclusion of a feature in one category of claims does not
imply a limitation to this category, but rather the feature may be
equally applicable to other claim categories, as appropriate. Where
a composition or process `comprises` one or more specified items or
steps, others can also be included. The invention also
contemplates, however, that the described composition or process
may be used without other items or steps and thus it includes the
recited composition or process `consisting of` or `consisting
essentially of` the recited items, materials or steps, as those
terms are commonly understood in patent law.
[0120] Terms and phrases used in this document, and variations
thereof, unless otherwise expressly stated, should be construed as
open ended as opposed to limiting. As examples of the foregoing:
the term "including" should be read to mean "including, without
limitation" or the like; the terms "example" or "some variations"
are used to provide exemplary instances of the item in discussion,
not an exhaustive or limiting list thereof; and adjectives such as
"conventional," "traditional," "normal," "standard," "known" and
terms of similar meaning should not be construed as limiting the
item described to a given time period or to an item available as of
a given time, but instead should be read to encompass conventional,
traditional, normal, or standard technologies that may be available
or known now or at any time in the future. Likewise, a group of
items linked with the conjunction "and" should not be read as
requiring that each and every one of those items be present in the
grouping, but rather should be read as "and/or" unless expressly
stated otherwise. Similarly, a group of items linked with the
conjunction "or" should not be read as requiring mutual exclusivity
among that group, but rather should also be read as "and/or" unless
expressly stated otherwise. Furthermore, although items, elements
or components of methods and compositions described herein may be
described or claimed in the singular, the plural is contemplated to
be within the scope thereof unless limitation to the singular is
explicitly stated. The presence of broadening words and phrases
such as "one or more," "at least," "but not limited to," "in some
variations," "in some non-limiting variations" or other like
phrases in some instances shall not be read to mean that the
narrower case is intended or required in instances where such
broadening phrases may be absent.
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